Bifeng Pan
Bifeng Pan
Bifeng Pan
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Nanomaterials-Mediated Transfer of<br />
siRNA Induced Apoptosis and<br />
Attenuated Tumor Cell Growth in Vitro<br />
Dr. <strong>Bifeng</strong> <strong>Pan</strong> & Prof. Daxiang Cui<br />
Department of Bio-Nano Science and Engineering Institute<br />
of Micro/nano Science and Technology Shanghai Jiao Tong<br />
University, P. R. China
Outline<br />
1. Nanomaterials Preparation:<br />
Dendrimer, Magnetic nanoparticles, Carbon nanotubes<br />
2. SiRNA –conjugated Nanoparticles Preparation<br />
3. Effects of SiRNA-conjugated Nanoparticles on Cancer<br />
cells<br />
4. Conclusion
Introduction<br />
Nanotechnology has tremendous potential in creating nanoparticle systems for<br />
targeted drug delivery.<br />
Nanoscale drug delivery system can deliver drugs to specific tissues in the body, and<br />
enhance drug penetration into cells, and improve drug activity.<br />
Cellular surface machinery and intracellular organelles operate at the nanolevel:<br />
regulate the actions of messenger molecules, maintain ionic stability, and<br />
manufacture a wide variety of crucial building blocks. Biochemical molecules ‘dock’<br />
into larger nanoscale structures (10-100 nm) to mediate specific functions, or are<br />
processed further through the active sites of receptors and enzymes.<br />
Nanoparticles are ideal for interacting on the nanoscale and enabling effective and<br />
selective therapeutics.<br />
We will focus on nanoparticle systems for Si RNA delivery with the goal of<br />
achieving ‘multi-targeted’ therapeutics.
Dendrimer<br />
Preparation procedure
Three dimensional<br />
structure<br />
Molecular Structure
Polyamidoamine<br />
(PAMAM)<br />
Dendrimer<br />
Chemical Structure
Dendrimer Coated Magnetite Nanoparticles<br />
Synthesis Procedure. (<strong>Bifeng</strong> <strong>Pan</strong> et al, J Colloid Interface Sci, 2005, 284:<br />
1-6)
TEM Characterization<br />
Magnetite nanoparticles without dendrimer coating<br />
(<strong>Bifeng</strong> <strong>Pan</strong> et al, J Colloid Interface Sci, 2005, 284: 1-6)
TEM photos<br />
G3.0 PAMAM dendrimer coated<br />
magnetite nanoparticles(<strong>Bifeng</strong> <strong>Pan</strong> et al, J<br />
Colloid Interface Sci, 2005, 284: 1-6)
TEM photos<br />
G5.0 PAMAM dendrimer coated<br />
magnetite nanoparticles(<strong>Bifeng</strong> <strong>Pan</strong> et al, J<br />
Colloid Interface Sci, 2005, 284: 1-6)
Size Distribution<br />
Diameter<br />
distribution of<br />
magnetite<br />
nanoparticles (<strong>Bifeng</strong><br />
<strong>Pan</strong> et al, J Colloid Interface<br />
Sci, 2005, 284: 1-6)
TGA analysis<br />
TGA curves of<br />
magnetite<br />
nanoparticles<br />
modified with<br />
aminosilane<br />
(G0) and with<br />
PAMAM<br />
dendrimers<br />
(G1–G5).<br />
(<strong>Bifeng</strong> <strong>Pan</strong> et al, J<br />
Colloid Interface Sci,<br />
2005, 284: 1-6)
FTIR analysis<br />
FT-IR spectra of (a) magnetite nanoparticles modified with APTS<br />
(G0), (b) G5 PAMAM dendrimer-modified magnetite nanoparticles, and<br />
(c) G5 PAMAM dendrimers. (<strong>Bifeng</strong> <strong>Pan</strong> et al, J Colloid Interface Sci, 2005,<br />
284: 1-6)
Zeta potential characterization<br />
Zeta-potential curves of magnetite nanoparticles modified with<br />
APTS (G0) and PAMAM dendrimers (G1–G5).<br />
(<strong>Bifeng</strong> <strong>Pan</strong> et al, J Colloid Interface Sci, 2005, 284: 1-6)
Preparation of dendrimer coated carbon nanotubes<br />
Growth of PAMAM dendrimer on the surface of carbon nanotubes<br />
<strong>Bifeng</strong> <strong>Pan</strong>, Daxiang Cui, et al. Nanotechnology 17 (2006) 2483–2489
TEM Characterization<br />
CNT without dendrimer modification
TEM photos<br />
Dendrimer coated CNT
TEM photos<br />
Dendrimer modified CNT
Dendrimer<br />
coated CNT<br />
TEM photos
UV-vis spectroscopy<br />
UV–vis spectra of (a) MWNT-NH2 and (b) dendrimer-modified CNT.
Raman spectroscopy<br />
Raman spectra (780 nm excitation) of (A) CNT-COOH and (B) dendrimercoated<br />
CNT
FT-IR spectroscopy<br />
FTIR spectrum of dendrimer-modified and uncoated CNT.
TGA analysis<br />
TGA thermograms of dendrimer coated CNTs
1 H NMR spectroscopy<br />
1 H NMR spectrum of dendrimer-modified CNT
Gene silence by small interference RNA<br />
Design Principle of siRNA
Principle of<br />
RNA silence<br />
by siRNA<br />
Schematic<br />
illustration of<br />
RNA silence
Survivin gene silence by siRNA<br />
5 steps of survivin gene RNA silence
Animal experiment<br />
Gene silence in vivo of siRNA by delivery vectors.
Survivin gene<br />
introduction (gene<br />
bank NM_001168)<br />
mRNA sequence<br />
1 cccagaaggc cgcggggggt ggaccgccta agagggcgtg cgctcccgac atgccccgcg<br />
61 gcgcgccatt aaccgccaga tttgaatcgc gggacccgtt ggcagaggtg gcggcggcgg<br />
121 catgggtgcc ccgacgttgc cccctgcctg gcagcccttt ctcaaggacc accgcatctc<br />
181 tacattcaag aactggccct tcttggaggg ctgcgcctgc accccggagc ggatggccga<br />
241 ggctggcttc atccactgcc ccactgagaa cgagccagac ttggcccagt gtttcttctg<br />
301 cttcaaggag ctggaaggct gggagccaga tgacgacccc atagaggaac ataaaaagca<br />
361 ttcgtccggt tgcgctttcc tttctgtcaa gaagcagttt gaagaattaa cccttggtga<br />
421 atttttgaaa ctggacagag aaagagccaa gaacaaaatt gcaaaggaaa ccaacaataa<br />
481 gaagaaagaa tttgaggaaa ctgcggagaa agtgcgccgt gccatcgagc agctggctgc<br />
541 catggattga ggcctctggc cggagctgcc tggtcccaga gtggctgcac cacttccagg<br />
601 gtttattccc tggtgccacc agccttcctg tgggcccctt agcaatgtct taggaaagga<br />
661 gatcaacatt ttcaaattag atgtttcaac tgtgctcttg ttttgtcttg aaagtggcac<br />
721 cagaggtgct tctgcctgtg cagcgggtgc tgctggtaac agtggctgct tctctctctc<br />
781 tctctctttt ttgggggctc atttttgctg ttttgattcc cgggcttacc aggtgagaag<br />
841 tgagggagga agaaggcagt gtcccttttg ctagagctga cagctttgtt cgcgtgggca<br />
901 gagccttcca cagtgaatgt gtctggacct catgttgttg aggctgtcac agtcctgagt<br />
961 gtggacttgg caggtgcctg ttgaatctga gctgcaggtt ccttatctgt cacacctgtg<br />
1021 cctcctcaga ggacagtttt tttgttgttg tgtttttttg tttttttttt tttggtagat<br />
1081 gcatgacttg tgtgtgatga gagaatggag acagagtccc tggctcctct actgtttaac<br />
1141 aacatggctt tcttattttg tttgaattgt taattcacag aatagcacaa actacaatta<br />
1201 aaactaagca caaagccatt ctaagtcatt ggggaaacgg ggtgaacttc aggtggatga<br />
1261 ggagacagaa tagagtgata ggaagcgtct ggcagatact ccttttgcca ctgctgtgtg<br />
1321 attagacagg cccagtgagc cgcggggcac atgctggccg ctcctccctc agaaaaaggc<br />
1381 agtggcctaa atccttttta aatgacttgg ctcgatgctg tgggggactg gctgggctgc<br />
1441 tgcaggccgt gtgtctgtca gcccaacctt cacatctgtc acgttctcca cacgggggag<br />
1501 agacgcagtc cgcccaggtc cccgctttct ttggaggcag cagctcccgc agggctgaag<br />
1561 tctggcgtaa gatgatggat ttgattcgcc ctcctccctg tcatagagct gcagggtgga<br />
1621 ttgttacagc ttcgctggaa acctctggag gtcatctcgg ctgttcctga gaaataaaaa<br />
1681 gcctgtcatt tcaaacactg ctgtggaccc tactgggttt ttaaaatatt gtcagttttt<br />
1741 catcgtcgtc cctagcctgc caacagccat ctgcccagac agccgcagtg aggatgagcg<br />
1801 tcctggcaga gacgcagttg tctctgggcg cttgccagag ccacgaaccc cagacctgtt<br />
1861 tgtatcatcc gggctccttc cgggcagaaa caactgaaaa tgcacttcag acccacttat<br />
1921 ttctgccaca tctgagtcgg cctgagatag acttttccct ctaaactggg agaatatcac<br />
1981 agtggttttt gttagcagaa aatgcactcc agcctctgta ctcatctaag ctgcttattt<br />
2041 ttgatatttg tgtcagtctg taaatggata cttcacttta ataactgttg cttagtaatt<br />
2101 ggctttgtag agaagctgga aaaaaatggt tttgtcttca actcctttgc atgccaggcg<br />
2161 gtgatgtgga tctcggcttc tgtgagcctg tgctgtgggc agggctgagc tggagccgcc<br />
2221 cctctcagcc cgcctgccac ggcctttcct taaaggccat ccttaaaacc agaccctcat<br />
2281 ggctaccagc acctgaaagc ttcctcgaca tctgttaata aagccgtagg cccttgtcta<br />
2341 agtgcaaccg cctagacttt ctttcagata catgtccaca tgtccatttt tcaggttctc<br />
2401 taagttggag tggagtctgg gaagggttgt gaatgaggct tctgggctat gggtgaggtt<br />
2461 ccaatggcag gttagagccc ctcgggccaa ctgccatcct ggaaagtaga gacagcagtg<br />
2521 cccgctgccc agaagagacc agcaagccaa actggagccc ccattgcagg ctgtcgccat<br />
2581 gtggaaagag taactcacaa ttgccaataa agtctcatgt ggttttatct aaaaaaaaaa<br />
2641 aaaaaaaaaa aaaaa
Design of shRNA plasmid express vector<br />
1. PGPU6/GFP/Neo-shNC<br />
2. PGPU6/GFP/Neo-shGAPDH<br />
3. PGPU6/GFP/Neo-shSurvivin51<br />
4. PGPU6/GFP/Neo-shSurvivin166<br />
5. PGPU6/GFP/Neo-shSurvivin261<br />
6. PGPU6/GFP/Neo-shSurvivin409
Survivin-51<br />
Target: GCAUCUCUACAUUCAAGAA<br />
Survivin-51 sense<br />
CACCGCATCTTCTACATTCAAGAATTCAAGAGATTCTTGAATGTAGAGATGCTTTTTT<br />
G<br />
Survivin-51 antisense<br />
GATCCAAAAAAGCATCTCTACATTCAAGAATCTCTTGAATTCTTGAATGTAGAGATG<br />
C<br />
5’ ACCGCATCTTCTACATTCAAGAATTCAAGAGATTCTTGAATGTAGAGATGCTTTTTTG 3’<br />
3’ CGTAGAGATGTAAGTTCTTAAGTTCTCTAAGAACTTACATCTCTACGAAAAAACCTAG 5’
Survivin-166<br />
Target: GGACCACCGCAUCUCUACA<br />
Survivin-166 sense<br />
CACCG GACCA CCGCA TCTCT ACATT CAAGA GATGT AGAGA<br />
TGCGG TGGTC CTTTT TTG<br />
Survivin-166 antisense<br />
GATCC AAAAA AGGAC CACCG CATCT CTACA TCTCT TGAAT<br />
GTAGA GATGC GGTGGTCC<br />
5’ CACCG GACCA CCGCA TCTCT ACATT CAAGA GATGT AGAGA TGCGG TGGTC CTTTT TTG<br />
3’ CCTGG TGGCG TAGAG ATGTA AGTTC TCTAC ATCTC TACGC CACCA GGAAA AAACC TAG 5’
Survivin 261<br />
Target: CUGUCAAGAAGCAGUUUGAdTdT<br />
Survivin 261 sense<br />
CACCGCTGTCAAGAAGCAGTTTGATTCAAGAGATCAAACTGCT<br />
TCTTGACAGTTTTTTTG<br />
Survivin 261 antisense<br />
GATCCAAAAAACTGTCAAGAAGCAGTTTGATCTCTTGAATCAA<br />
ACTGCTTCTTGACAGC<br />
5’ CACCGCTGTCAAGAAGCAGTTTGATTCAAGAGATCAAACTGCTTCTTGACAGTTTTTTTG 3’<br />
3’ CCACAGTTCTTCGTCAAACTAACTTCTCTAGT TTGACGAAG AACTGTCAA AAAACCTAG 5’
Survivin 409<br />
Target: GCUGGCUGCCAUGGAUUGA<br />
Survivin-409 sense<br />
CACCGCTGGCTGCCATGGATTGATTCAAGAGATCAATCCATG<br />
GCAGCCAGCTTTTTTG<br />
Survivin-409 antisense<br />
GATCCAAAAAAGCTGGCTGCCATGGATTGATCTCTTGAATCAA<br />
TCCATGGCAGCCAGC<br />
5’ CACCGCTGGCTGCCATGGATTGATTCAAGAGATCAATCCATGGCAGCCAGCTTTTTTG 3’<br />
3’ CGTAGA GATGT AAGT TCTT AAGT TCTCTAAGAACTTACATCTCTACGAAAAAACCTAG5’
Negative Control<br />
Sense<br />
5’CACCGTTCTCCGAACGTGTCACGTCAAGAGATTACGTGACA<br />
CGTTCGGAGAATTTTTTG-3’<br />
Anti-Sense<br />
5’GATCCAAAAAATTCTCCGAACGTGTCACGTAATCTCTTGACG<br />
TGACACGTTCGGAGAAC3’<br />
5’ CACCGTTCTCCGAACGTGTCACGTCAAGAGATTACGTGACACGTTCGGAGAATTTTTTG 3’<br />
3’ CAAGAGGCTTGCA CAGTGC AGTTCTCTAATGCACTGTGCA AGCCTCTAAAAAACCTAG 5’
shGAPDH<br />
Sense<br />
5’CACCGTATGACAACAGCCTCAAGTTCAAGAGACTTGAGGCT<br />
GTTGTCATACTTTTTTG3’<br />
Antisense<br />
5’GATCCAAAAAAGTATGACAACAGCCTCAAGTCTCTTGAACTT<br />
GAGGCTGTTGTCATAC3’<br />
5’CACCGTATGACAACAGCCTCAAGTTCAAGAGACTTGAGGCTGTTGTCATACTTTTTTG3’<br />
3’CATACTGTTGTCGGAGTTCAAGTTCTCTGAACTCCGACAACAGTATGAAAAAACCTAG5’
shRNA plasmid vector<br />
PGPU6/GFP/Neo-shSurvivin166
Design of siRNA against survivin<br />
Survivin-51<br />
Sense: 5’-GCAUCUCUACAUUCAAGAAdTdT-3’<br />
Antisense: 5’-UUCUUGAAUGUAGAGAUGCdGdG-3’<br />
Survivin-166<br />
Sense: 5’-GGACCACCGCAUCUCUACAdTdT-3’<br />
Antisense: 5’-UGUAGAGAUGCGGUGGUCCdTdT-3’<br />
Survivin-261<br />
Sense: 5’-CUGUCAAGAAGCAGUUUGAdTdT-3’<br />
Antisense: 5’-UCAAACUGCUUCUUGACAGdAdA-3’<br />
Survivin-409<br />
Sense: 5’-GCUGGCUGCCAUGGAUUGAdTdT-3’<br />
Antisense: 5’-UCAAUCCAUGGCAGCCAGCdTdG-3’<br />
GAPDH Positive control<br />
Sense: 5’-GUAUGACAACAGCCUCAAGTT-3’<br />
Antisense: 5’-CUUGAGGCUGUUGUCAUACTT-3’<br />
Negative control<br />
Sense: 5’-UUCUCCGAACGUGUCACGUTT-3’<br />
Antisense: 5’-ACGUGACACGUUCGGAGAATT-3’<br />
Negative control FAM<br />
Sense: 5’-UUCUCCGAACGUGUCACGUTT-3’<br />
Antisense: 5’-ACGUGACACGUUCGGAGAATT-3’
Interaction between<br />
shRNA, siRNA and<br />
Nanomaterials<br />
Gel electrophoresis
Zeta potential analysis<br />
Electrostatic interaction between nanomaterials and siRNA
Schematic illustration of interaction<br />
between nanomaterials and siRNA<br />
Electrostatic interaction
Cancer cell culture<br />
1. Breast cancer cell line: MDA-MB-435-S<br />
2. Breast cancer cell line: MDA-MB-231<br />
3. Breast cancer cell line: MCF-7<br />
4. Human liver cancer cell line: HepG2<br />
5. Mouse fibroblast cell line: L-929
Optical microscopy observation<br />
Photo of MCF-7 cells
Optical microscopy observation<br />
MCF-7 Cells Apoptosis Induced by<br />
Nanomaterials-shRNA complex
Cell growth inhibition by MTT assay<br />
MCF-7 cells apaptosis anaylsis
Western Blotting<br />
MCF-7 Cells<br />
Primary antibody: anti-survivin polyclonal<br />
antibody<br />
The secondary antibody: horseradish<br />
peroxidase-conjugated goat anti-rabbit IgG<br />
GAPDH<br />
Survivin
RT-PCR analysis<br />
The survivin cDNA was amplified with the forward primer 5'-GGG GGA CTG<br />
GCT GGG CTG CT-3'(1422-1441)and reverse primer 5'-TGG GGT TCG<br />
TGG CTC TGG CAA-3'(1832-1852), resulting in a fragment of 430 bp
Confocal microscopy observation<br />
MCF-7 cell incubation in the presence of<br />
FAM labelled shRNA Without adding<br />
nanomaterials
FAM labelled shRNA With nanomaterials In<br />
MCF-7 cells by Confocal Micorscopy
CNTs inside MCF -7 cells by HRTEM
Influence of nanoparticles conjugated<br />
siRNA on cellular cycles by FCM
Conclusions<br />
� Nanotechnology-based advanced materials are<br />
rapidly expanding development of better<br />
medicines.<br />
� siRNA –conjugated Nanoparticles can enter into<br />
tumor cells and inhibit tumor cells growth .<br />
� Nanoscale delivery system creates a new<br />
generation of ‘targeted’ therapeutics which can<br />
offer multiple levels of selectivity.
Department of Bio-Nano Bio Nano Science and<br />
Technology, Shanghai Jiao Tong University
Shanghai Jiao Tong<br />
University<br />
http://www.sjtu.edu.cn<br />
http://mnri.sjtu.edu.cn/
Thanks<br />
For your attention!
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